It is a well known and well documented fact that the demand for blood supplies for administration to patients undergoing surgery and other emergency medical procedures has increased very rapidly over the past 30 years or so. The demand often exceeds the supplies available from human donors. Even larger volumes of blood would be used if it were readily available. Elective surgery is often postponed because of shortages of blood. Sophisticated medical techniques such as organ transplants continue to become more successful and more common so that the amounts of blood required continues to increase. Extracorporeal techniques require large quantities of blood, mostly for temporary use. There is therefore a need to develop blood supplies which are available. The need exists not only in areas where advanced medical techniques are practiced, but also in underdeveloped areas of the world where expensive facilities for blood banking are not available.
The use of whole blood for transfusions has several known disadvantages. To avoid an antigenic reaction, the recipient's blood must be accurately typed and matched to compatible blood from a donor, necessitating excess stored units and the loss of valuable time in emergency situations. It can only be transfused in hospitals rather than emergency vehicles at the site of a trauma. Currently, whole blood can be stored at 4.degree. C. for no longer than three weeks before a considerable fraction of the red blood cells become osmotically fragile and non-viable. Frozen cells must be washed free of glycerol, which is expensive and time consuming, and these cells are also somewhat osmotically fragile. Also, the risk of transmitting disease by transfused blood is quite high, most notably non A/non B hepatitis, parasites and AIDS.
A blood substitute capable of more than just fluid replacement has been actively sought by researchers around the world for some 15 years and in Japan perfluorinated hydrocarbons are currently being used in this context. Oxygen is very soluble in these compounds, but ambient oxygen is not sufficient to satisfactorily improve the oxygen carrying capacity, necessitating an oxygen tent which is unsuitable for many emergency situations, especially combat emergencies. It has also been declared unsuitable for clinical trials in this country due to other complications. To avoid these difficulaties, hemoglobin has been suggested and used as a blood substitute.
Use of hemoglobin solutions has the advantage as compared with use of whole blood, that blood typing would not have to be undertaken. Such solutions therefore could be given to a patient in an emergency without taking the time to type and cross-match the blood. Blood types are genetically determined and are the result of specific antigens present on the surface of the red blood cells (RBCs). The hemoglobin within the cells does not exhibit a blood type once separated from the cell membranes or stroma. Moreover, hemoglobin is a much easier material to store than whole blood, and does not deteriorate as quickly. Stocks of blood have to be discarded after a relatively short period of time. Hemoglobin can be isolated from blood and frozen so that it can be stored for a much longer period of time. Use of hemoglobin solutions instead of whole blood thus would have significant advantages and would tend to alleviate problems of lack of supply of whole blood, particularly lack of supply of blood of specific types.
However, hemoglobin is rapidly excreted by the kidneys into the urine and some resultant renal dysfunction has been observed. Frequent massive transfusions of hemoblobin solution, if employed to balance the high rate of excretion, would certainly pose a hazard to patients with pre-existing renal disease. It has been reported that the circulation half-life, defined as the time for disappearance of half of the hemoglobin administered in a solution by transfusion, is only one and one-half hours in monkeys.
Therefore, there have been efforts to encapsulate stroma-free hemoglobin in an antigen-free encapsulant which would allow for adequate oxygenation of the hemoglobin, prevent renal excretion of the hemoglobin, and insure ample circulation half-time of the hemoglobin. The principal difficulty with these efforts heretofore has been the inability for preparing enough product with sufficient 0.sub.2 --carrying capacity for large-scale animal testing.
The present synthetic red cell concept actually dates from 1964 when T. M. S. Chang first encapsulated hemoglobin in collodion or Nylon membranes (T. M. S. Chang), Science 146:524 (1964)). Crosslinked hemoglobin used as a membrane (T. A. Davis, W. J. Asher, and H. W. Wallace, Appl. Biochem. Biotech. 10:123 (1984)), (M. C. Levy, P. Rambourg, J. Levy, and G. Potron, J. Pharmaceut. Sci. 71:759 (1982)) and other polymeric membranes (M. Arakawa, A. Kato, and T. Kondo, Appl. Biochem. Biotech. 10:143 (1984)), J. A. Hayward, D. M. Levine, L. Neufeld, S. R. Simon, D. S. Johnston, and D. Chapman, FEBS Letters 187:261 (1985)), M. Ndong-Hkoume, P. Labrude, J. C. Humbert, B. Teisseire, and C. Vigneron, Annales Pharmaceut. Franc 39:247 (1981)), now are being investigated as well. Oxidation of the hemoglobin has been a complication with these methods so far, though they all hold promise. Another version is the incorporation of iron-porphyrin derivatives in the membrane of liposomes rather than in the globin protein, such that the "cell" membrane rather than the aqueous interior serves as the 0.sub.2 carrying site (E. Tsuchida, H. Nishide, M. Yuasa, and M. Sekine, Bull. Chem. Soc. Japan, 57:776 (1984)).
U.S. Pat. No. 4,133,874 to Miller et al., incorporated herein by reference, describes lipid-encapsulated hemoglobin cells. One embodiment contains lecithin ex ovo, cholesterol, and phosphatidic acid in a 15:10:1 molar ratio. Another embodiment contains lecithin ex ovo, cholesterol and phosphatidylserine in a 9:7:1 ratio. The method described by Miller et al. has not been amenable to scale up (Business Week, 17 June 1985 page 149). Moreover, the sizes of the hemoglobin "cells" obtained were not uniform, ranging from 0.1 to 10 microns, potentially inhibiting proper circulation. In addition, the vigorous stirring or ultrasonic energy required by Miller et al. tends to result in some damage to the encapsulated hemoglobin. Further, the Miller "cells" lock a well-defined structure and are multilayered. A method described by Gaber et al. for liposome encapsulated hemoglobin, patent application Ser. No. 508,692 (now abandoned), had the advantage of a uniform "cell" size, but depends on successive extrusions through membranous filters which yield a final product of approximately 1 ml/sq. in. of filter. The largest extrusion chambers available are not adequate to produce a batch size of one liter of liposomes by this method as well as high encapsulation efficiency. The method also used synthetic phospholipids which had the advantage of purity, but are excessively expensive. The method of Hunt et al., U.S. Pat. No. 4,425,334 involves six steps in the encapsulation alone and has proven very difficult to scale up (Science 230, p. 1165, 1985). All three of the above mentioned methods use different phospholipids, yet the circulation half-life as measured in mice is only about 4-5 hours in each case, substantially less than desirable for a blood replacement.